Conflicting opinions for the role of CD4+ T lymphocytes in CBPP have been published [5, 9]. These are based on the presence or absence of a correlation between high numbers of IFN-γ-secreting CD4+ T cells with mild disease outcome. As these studies never tested cause and effect, the aim of this study was to whether M. mycoides subsp. mycoides-specific CD4+ T lymphocytes can alter the course of primary CBPP infection. We compared two groups of 10 animals, each infected with M. mycoides subsp. mycoides strain Afadé, one depleted for CD4+ T lymphocytes six days pi and the other left untreated. Clinical parameters such as fever and cough, as well as post mortem results were recorded. Ten animals per experimental group were chosen based on statistical considerations and on the assumption that the depleted group would show a dramatic increase in disease severity if the CD4+ T cells were involved in control of the infection.
A total of 118 mg/animal of the CD4+-specific monoclonal antibody IL-A11 was administered intravenously in five increasing doses, to achieve in vivo depletion of CD4+ T cells. Depletion was successful as confirmed by flow cytometry showing no CD4+ cells one day after treatment in peripheral blood. From previous depletion experiments, it was assumed that CD4+ T cells were also completely removed from lymphoid tissues, with exception of the thymus . Naïve CD4+ cells could be detected in low numbers about one week later. The CD4+ T cell numbers remained below 25% of normal throughout the entire experiment. The data shows that removal of CD4+ T cells did not result in clear differences between both groups. No significant differences were found in fever or the frequency of coughing, both good correlates of clinical CBPP severity. Additionally, the number and size of lung lesions did not differ between both groups. The difference observed in mortality (3/10 versus 1/10), or more correctly in the number of animals with symptoms that necessitated euthanasia, was statistically significant (p < 0.07), though low and could be interpreted as being caused by loss of a protective response. The numbers demonstrate that the effect of depletion on mortality is minor.
In order to ensure that the depletion of a T cell population did not increase the presence of unrelated pathogens that could confound the pathology, the presence of a number of possible bacteria and viruses were monitored before and after infection. In related mycoplasma, such as M. bovis and M. hyopneumoniae, co-infections with other pathogens which may contribute to lung pathology have been observed. Evidence for the presence of several viruses was detected in our study, however, no major differences were observed between the two groups. Interestingly we observed a substantial increase in antibody against BHV-4 after infection in both groups, which suggests an immuno-compromised state in the animals during clinical CBPP. We speculate that additional pathogens may affect CBPP infected animals, which could result in even more acute clinical symptoms.
One concern is that low numbers of CD4+ T cells can mediate a protective function. This is difficult to accept as low numbers of IFN-γ-secreting CD4+ T cells in acute animals were not sufficient to prevent acute disease . Another concern is that the role of CD4+ T cells is compensated by another cell type in treated animals. This cannot be disproven, but is unlikely. The CD4+ population contains cells that provide regulatory functions, such as helper and suppressor tasks, which other cell types cannot do. Also, it is unlikely that depletion of peripheral IFN-γ-secreting CD4+ T cells can trigger a compensating mechanism, while very low numbers (in acutely infected cattle) cannot. Furthermore, no compensation in number of other populations was observed in previous CD4+ depletion experiments .
A reduction in antibodies in the depleted animals measured by the complement fixation test was not observed, as would be expected after eliminating a large portion of T cell help. A reduction in antibody production has been observed in previous CD4+ depletion experiments in cattle or sheep [20–22]. It is possible that antibody production had begun prior to the depletion treatment. This seems unlikely as antibodies were first detected in blood by day nine and the kinetics of the antibody responses after that time did not statistically differ between treated and control groups. Another explanation is that anti-mycoplasma antibodies might be produced without T cell help. Such T cell-independent antibodies have been described in trypanosome infections, where antibody titres in infected nu/nu mice were only slightly lower than in normal mice . Mycoplasma express high amounts of carbohydrate on their surface , which might form repetitive epitopes that need less T cell help to induce specific antibodies.
One final concern is the timing of depletion. We depleted six dpi, since previous experimental infections carried out in Kenya and Namibia suggested that the peak of clinical symptoms occurred around 2-3 weeks pi [9, 25] and previous depletion experiments suggested complete elimination of T cells for one to two weeks after treatment. We succeeded in removing the CD4+ T cells before the peak of clinical symptoms, but one can argue that a protective CD4 response acted prior to the depletion, or after CD4+ T cells returned. If the protective immune response in animals with mild symptoms of disease was driven by high numbers of IFN-γ-secreting CD4+ T cells, we would not expect such cells to appear early, as demonstrated in the previous studies [7, 8]. Also, while naïve CD4+ T cells appeared from the thymus one week after treatment, one would expect that a hypothetical, protective CD4+ T cell response would be seriously delayed in the depleted animals, leading to severe disease.
If CD4+ T cells do not provide the protective response in a primary infection, what defines whether an animal develops severe (acute) or a less severe (chronic) diseaseż Since there is no direct correlation between antibody titres and disease severity, it is difficult to ascribe a protective role to high antibody titers. Therefore, we assume that innate responses may play an important role. Of course, our data do not rule out a protective role for CD4+ T cells in combination with antibody production after vaccination or during a secondary infection.
CBPP is a fatal disease particularly in naïve herds where up to > 30% of animals may die , indicating that during a primary infection the induced responses are not sufficient or appear too late to adequately control the disease. Following this reasoning, it is not surprising that acquired responses driven by CD4+ T cells only play a minor role in control of primary CBPP infection as previously speculated . In our opinion future research efforts towards the identification of protective immune responses should be focused on an infection model employing immune animals.